c-MYC is among the most commonly overexpressed genes in smoking-related cancers, including those of the head and neck. Ectopic expression of c-MYC is one of only five distinct molecular alterations needed for transformation of primary oral keratinocytes.Yet, the molecular mechanisms by which c-MYC induces cellular transformation remain poorly understood. The dyskeratosis congenita 1 (DKC1) gene is a component of two molecular pathways that are regulated by c-MYC. Our preliminary data described herein indicate that (1) DKC1 is a direct and conserved transcriptional target of c-MYC, (2) DKC1 is frequently overexpressed in oral squamous cell carcinomas;and c-MYC and DKC1 expression correlate in a subset of tumors and cell lines, and (3) DKC1-mutant cells exhibit premature senescence and cell death. This has led us to hypothesize that DKC1 plays an important role in cancer formation;and DKC1 is an essential effector of c-MYC-mediated tumorigenesis. The objectives of this R03 proposal are: (1) Determine the role of DKC1 in c-MYC-induced cellular proliferation and transformation;and (2) Determine if overexpression of DKC1 can recapitulate c-MYC function in susceptible cells. Overexpression of c-MYC stimulates proliferation and induces cellular transformation. In contrast, c-Myc-null cells grow slowly and exhibit delayed progression through G2 phase. We propose that c-MYC requires DKC1 to sustain cellular proliferation and induce transformation. To test our hypotheses in Aim 1, we will overexpress c-MYC in DKC1-mutant or wild-type human lymphoblasts. In another experiment, we will use short hairpin RNA to silence DKC1 in primary oral keratinocytes that express an inducible c-MYC-estrogen receptor (MYC-ER) fusion protein. We will then measure senescence-associated-2- galactosidase production;and use standard assays and flow cytometry to investigate changes in proliferation and cell cycle distribution of the keratinocytes and lymphoblasts. Then, we will knockdown Dkc1 in wild-type Rat 1a-MYC-ER fibroblasts;and investigate the ability of MYC-ER to stimulate anchorage-independent growth in soft agar. In Aim 2, we will investigate the effects of DKC1 overexpression in c-Myc-null and wild type Rat 1a cells. We propose that ectopic expression of DKC1 will alleviate the G2 cell cycle defect and increase the proliferation rate of the c-Myc-null cells. We further hypothesize that DKC1 overexpression will also increase proliferation and induce transformation of the wild-type cells. To test our hypotheses, we will overexpress DKC1 in the c-Myc-null cells, and then measure the effects on cell proliferation and DNA synthesis. Flow cytometry will be used to assess changes in cell cycle distribution;and, in particular, the status of the cells in G2. We will then overexpress DKC1 in wild-type cells and determine the effect on anchorage-independent growth. The experiments outlined here investigate the effects of the loss and gain of DKC1 function on cell proliferation and transformation. By taking this approach, the proposed studies will lead to greater insight into the role of DKC1 in cancer development;and as an effector of c-MYC-mediated tumorigenesis. Narrative: Elucidating a significant role for DKC1 in tumorigenesis may lead to new avenues of study into the mechanisms that underlie the development of smoking-related cancers. Identifying a role for DKC1 may also lead to the development of new therapeutic strategies, thereby potentially increasing the quality of life and the long-term survival rate of affected individuals with smoking-associated cancers. Identifying DKC1 as an essential effector of c-MYC-mediated tumorigenesis may also lead to new targeted therapies for c-MYC-positive tumors.